The mechanisms by which antibody genes are regulated in a cell type- specific manner in response to cytokines and antigenic stimuli is poorly understood. We have developed a model system to study the mechanisms by which the antigen, phosphocholine, and the cytokine, interleukin-5, influence B cell differentiation and have found that IL-5 + antigen caused increased transcription of mu immunoglobulin genes. Several DNA- binding proteins that may be important in mitogen-stimulated or basal immunoglobulin transcription have been shown to bind within 200 base pairs of the transcription start site. IL-5+ antigen induced increases in B cell-specific mobility-shifted protein-DNA complexes that bound to A+T rich sequences 200 to 500 base pairs 5' of the transcription start site. These protein-DNA complexes probably contain more than one protein. In the proposed studies, these proteins will be characterized and their functions will be investigated. The broad objective of this research is to identify some of the regulatory events that occur in B cells in response to IL-5+ antigen. The specific goals are: (1) to determine the molecular basis for the B cell-specific mobility of the previously identified IL-5+ antigen inducible DNA-protein complexes, (2) to determine whether the proteins in this complex regulate transcription by functioning as topoisomerases, DNA-bending proteins, or components of the nuclear matrix, and (3) to test whether these proteins are involved in maintaining B cell-specific immunoglobulin transcription. Proteins will be further characterized using UV crosslinking and mobility shift assays. Total understanding of the role these proteins play in transcription may require in vitro systems. Therefore, individual proteins will be enriched by conventional methods and purified with affinity columns. Genes encoding proteins that appear to be important in the response to IL-5+ antigen will be cloned using conventional and/or expression libraries. Preliminary data indicates that protein complexes from T cell extracts also bind to these sequences, and that these complexes contain some proteins different than those found in the B cell complexes. Other proteins appear to be found in both B and T cell extracts. To examine whether the proteins that are unique to B cells play a role in the cell type-specific expression of immunoglobulin, T cell transfectants containing cloned B cell-specific genes will be produced and examined for their ability to express and regulate B cell-specific genes. These studies should provide insight into mechanisms by which proteins regulate increased transcription in a cell type-specific manner, and may lead to the identification of other important B cell proteins required for intracellular signaling in response to IL-5 and antigen.